regulatory regions in the rat lactase-phlorizin hydrolase gene that control cell-specific expression

Regulatory Regions in the Rat Lactase-Phlorizin HydrolaseGene that Control Cell-Specific Expression

*‡Menno Verhave, *†‡Stephen D. Krasinski, *Sara I. Christian, *Sandrijn Van Schaik,*Gijs R. van Den Brink, *Edwina M. H. Doting, *Saskia M. Maas, *Katja C. Wolthers,

*‡Richard J. Grand, and *‡Robert K. Montgomery

*Division of Pediatric Gastroenterology and Nutrition, Department of Pediatrics, The Floating Hospital for Children, NewEngland Medical Center Hospitals; Tufts University School of Medicine and the Center for Gastroenterology Research on

Absorptive and Secretory Processes, Boston, Boston Massachusetts; †Tufts University School of Nutrition Science and Policy,Medford Massachusetts; ‡Current affiliation: Childrens Hospital, Boston, Massachusetts, U.S.A.

ABSTRACTObjectives: Lactase-phlorizin hydrolase (LPH) is anenterocyte-specific gene whose expression has been well-characterized, not only developmentally but also along thecrypt-villus axis and along the length of the small bowel. Pre-vious studies from the authors’ laboratory have demonstratedthat 2 kb of the 5�-flanking region of the rat LPH gene controlthe correct tissue, cell, and crypt-villus expression in transgenicanimals.Methods: To examine further the regulation conferred by thisregion, protein-DNA interactions were studied using DNase Ifootprint analyses in LPH-expressing and nonexpressing celllines. Functional delineation of this 5�-flanking sequence wasperformed using deletion analysis in transient transfection as-says.Results: Studies revealed a generally positive activity between

−74 and −37 bp, a cell-specific negative region between −210and −95 bp, and additional elements further toward the 5� ter-minus that conferred a highly cell-specific response in reporteractivity. Computer analysis of distal regions encompassingidentified footprints revealed potential binding sites for variousintestinal transcription factors. Co-transfection and electromo-bility shift assay experiments indicated binding of HNF3� atthree sites relevant to LPH expression.Conclusions: The data demonstrate that the cell specificity ofLPH gene expression depends upon both positive and negativeinteractions among elements in the first 2 kb of the LPH 5�-flanking region. JPGN 39:275–285, 2004. Key Words: De-velopment—Gene expression—Lactase-phlorizin hydrolase—Small intestine—Transcription factors. © 2004 LippincottWilliams & Wilkins

Lactase-phlorizin hydrolase (LPH) hydrolyzes lactose,the principal carbohydrate in milk, and is therefore es-sential to the nutrition of the newborn mammal. LPH isa microvillus membrane disaccharidase expressed onlyin villus enterocytes of the small intestine. In most suck-

ling mammals, LPH expression is maximal in the jeju-num and proximal ileum and lower in the proximal du-odenum and distal ileum (1). The developmental patternof lactase activity and protein abundance in rats (2–4)and the genetic pattern in adult humans (5) vary directlywith the abundance of LPH mRNA (6–11). The proximalto distal (horizontal) small bowel distribution and thedevelopmental changes of LPH expression in rats aretranscriptionally regulated (3). Thus, although posttran-scriptional events modulate LPH expression (12–14), itsmajor regulation probably occurs at the level of genetranscription.

The mechanisms controlling transcription of the LPHgene are now being determined. Experiments in trans-genic mice have indicated that elements responsible fortissue, cell, and crypt-villus specificity are contained inthe 5�-flanking regions of the rat (−2,038 to +15) and pig(−825 to −17) LPH genes (15–17). The high degree ofsequence homology in the proximal promoters of rat,

Received August 11, 2003; revised January 23, 2004; accepted Janu-ary 27, 2004.

Address correspondence and reprint requests to Dr. Robert K. Mont-gomery, GI Research, Enders 1220, Children’s Hospital, 300 Long-wood Avenue, Boston, MA 02115 U.S.A (e-mail: robert.montgomery@childrens.harvard.edu).

Supported by National Institutes of Health (NIH) Research GrantR37 DK-32658; the Center for Gastroenterology Research on Absorp-tive and Secretory Processes, NIH Digestive Diseases Core CenterGrant P30 DK34928; NIH Clinical Investigator Award K08 DK-02182(MY); American Gastroenterological Association Industry ScholarAward (MY); and grants from the Charles H. Hood Foundation (SDK)and the March of Dimes Birth Defects Foundation (SDK); and a Re-search Fellowship Exchange Program, Glaxo, The Netherlands (SvS,GvdB, EMHD, SMM, KCW).

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mouse, pig, and human LPH genes (70% identity in thefirst 100 bp of 5�-flanking sequence of all four), whichcontain consensus binding sites for the same transcrip-tion factors, suggest that there are similar regulatorymechanisms. Cdx-2 (14,16,18), GATA-4,5,6 (19,20),HNF-� and � (21), C/EBP� (21,22), HNF-3 (21), andHOXC11 (23) are all probable modulators of LPH geneexpression, but their role in the control of cell-specificgene expression is not yet clear. Although the high levelof sequence conservation in the proximal promoter of theLPH genes analyzed to date suggests it contains impor-tant functions, recent transgenic experiments indicatethat additional critical regulatory elements are located inthe more distal 5�-flanking region (17).

To define elements regulating cell-specific LPH ex-pression, we analyzed the 5�-flanking sequence of the ratLPH gene for protein-DNA binding sites using cells withand without LPH expression. DNase I footprint analysesof the rat gene revealed protein-DNA interactions at mul-tiple sites. Deletion analysis using transient transfectionexperiments indicated several elements that may contrib-ute to the cell-specific expression of LPH.

METHODS

Chemicals and Reagents

All chemicals were purchased from Sigma Chemical Com-pany (St. Louis, MO, U.S.A.), Gibco-BRL (Gaithersburg, MD,U.S.A.) or Fischer Scientific (Fair Lawn, NJ, U.S.A.) unlessindicated otherwise. Enzymes were purchased from Gibco-BRL, Promega Biotec (Madison, WI, U.S.A.) or PharmaciaBiotech (Piscataway, NJ, U.S.A.) unless indicated otherwise.Radioactive nucleotides were purchased from DuPont-New En-gland Nuclear (Boston, MA, U.S.A.) and [14C]-chloram-phenicol was purchased from Amersham Life Science (Arling-ton Heights, IL, U.S.A.).

Scanning DNase I Footprint Assays

To localize protein-DNA interactions in large (>200 bp) re-gions of the 5�-flanking sequence, scanning DNase I footprintassays were performed as described (24) with modifications.DNA fragments of up to 800 bp containing the region of in-terest were isolated by restriction digestion or by polymerasechain reaction amplification. The fragments were labeled usingpolynucleotide kinase and [32P]-dATP (37°C, 1 hour), digestedwith an enzyme to remove the label from one end, and purifiedby centrifugation through a G-50 column (5 Prime-3 Prime,Inc., Boulder, CO, U.S.A.) followed by extraction from agarosegels. The purified double-stranded probes (5 fmol) were incu-bated on ice for 10 minutes in 20 �L volumes with Caco-2 orHep-G2 nuclear extracts (75–115 �g), 2 �g poly (dI-dC):poly(dI:dC), and 10% polyvinyl alcohol in footprint buffer (250mM HEPES, pH 7.6, 340 mM KCl). Nuclear extracts wereprepared as described (25). The probes were then digested withDNase I (Worthington) for 1 to 2 minutes at 37°C. DNasereactions were terminated by addition of a stop solution con-taining 20 mM Tris-Cl, 20 mM EDTA, 250 mM NaCl, 0.5%

SDS, 4 �g/mL salmon sperm DNA, and 10 mg/mL proteinaseK. After incubation at 65°C for 15 minutes, the DNA was PICextracted, ethanol precipitated, and separated electrophoretical-ly in 6% denaturing polyacrylamide gels. A Maxam-GilbertG-sequence ladder was used as a size marker. Controls werefootprint assays without protein or with bovine serum albumin(BSA).

Construction of LPH-bGH Fusion Genes

To identify functionally active regions in the rat LPH 5�-flanking sequence that directed cell-specific expression, a se-ries of deletions of these sequences were fused 5� to the humangrowth hormone (hGH) reporter gene. Rat LPH 5�-flankingsequence was derived from a previously characterized rat ge-nomic subclone that included 2,038 bp of upstream sequence(15,26). Internal restriction sites or polymerase chain reactionswere used to make all deletions. An additional 2 kb of rat LPH5�-flanking sequence, isolated from the original clone (26), wasfused to r2038GH, resulting in a 4-kb rat LPH-hGH construct.All constructs were confirmed by sequencing.

Transient Transfection

To characterize the activity of the deletion constructs, tran-sient transfection assays in both LPH-expressing Caco-2 andLPH-nonexpressing Hep-G2, and in some experiments HeLa,cells were performed. The Caco-2 cells used in these experi-ments have been previously characterized by van Beers et al.(27).

All cells were transfected just before or at confluence (90%–100%) using 5.0 �g/kb of the LPH-hGH constructs and 1.0 �gof RSV-CAT as an internal control. pBluescript II KS+ (Strata-gene) was added as a carrier (total DNA/transfection � 50 �g).Transfections were performed by electroporation (Pulse Con-troller II, Bio-Rad, Hercules, CA, U.S.A.) at 300 volts and 950�F in buffer (240 mM KCl, 0.3 mM CaCl2, 20 mMHPO4/KH2PO4 pH 7.6, 50 mM HEPES pH 7.6, 4 mM EGTApH 7.6, 10 mM MgCl2 supplemented with fresh 3.08 mg/mLglutathione). After transfection, Caco-2 cells were replated intriplicate at 75% confluence and harvested 5 days after reach-ing confluence (6 days after transfection). The time of harvestcorresponds to the time of maximal endogenous lactase expres-sion (27) (Verhave, unpublished data). Transfected Hep-G2and HeLa cells were replated in duplicate at 90% confluenceand harvested 2 days later. All plates were confluent at the timeof harvest.

The amount of hGH secreted into the media in 24 hours wasused as an indicator of transcriptional activity. Thus, 24 hoursafter the last media change, media were collected, and theconcentration of hGH was measured using an [125I] immuno-assay kit (Allegro hGH, Nichols Institute, San Juan Capistrano,CA, U.S.A.). To analyze CAT activity, cell lysates were pre-pared by freeze-thaw cycling, and the protein concentration ofthe supernatant was determined by a Coomassie protein assay(Pierce, Rockford, IL, U.S.A.). CAT activity was measured incell lysates by thin layer chromatography as described (28). AllhGH levels were divided by CAT activity/microgram protein tocorrect for transfection efficiency. hGH/CAT activity of a pro-moterless growth hormone vector was subtracted from all otherconstructs to correct for background. All activity was expressed

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relative to an internal positive control, pXGH5, a constitutivelyactive metallothionine-I promoter fused to the hGH gene. Datafrom three replicate cultures were averaged to give n � 1. Alldata are at least n � 3, or more, as indicated.

Co-transfection Analysis

Previously characterized expression vectors for C/EBP�,�and � (29) and for HNF3� and � (30) were used in co-transfection experiments. A truncated 80 bp RSV promoter wasfused to hGH (designated RSVde) and used as a control in theco-transfection experiments.

Electromobility Shift Assay (EMSA)

Oligonucleotides encompassing the footprinted regions des-ignated LFP4, LFP7, LFP11, and LFP12 were synthesized bythe Core Center of the Tufts University Department of Physi-ology.

Nuclear proteins prepared as described were used. Assayswere performed as described (31). For supershift assays of theputative HNF3 binding sites, polyclonal antibodies againstHNF3� and HNF3� (generously provided by Dr. Robert Costa,University of Illinois College of Medicine, Chicago, IL,U.S.A.) were added subsequent to incubation of probe andprotein, and incubation continued for an additional 30 minutes.The antibodies were raised against purified HNF3� andHNF3� (32). Antibodies against C/EBP and HNF6 were pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA,U.S.A.).

Sequence and Data Analysis

Homology analyses were performed using the Genetic Com-puter Group (GCG) Software package (33). Putative transcrip-tion factor binding sites were identified using the MatInspectorprogram that uses the Transfac transcription factor database(34). Data in transient transfection assays were expressed asmean ± SD.

RESULTS

DNAse-I Footprint Analysis

To identify potential cell-specific elements in the ratLPH gene, a series of footprint assays were performedusing Caco-2 and Hep-G2 nuclear extracts. The probesused in the assays of the first 1.0 kb of rat LPH 5�-flanking sequence are indicated in Figure 1A. A similaranalysis was performed on the more distal region out to−2,038 bp. A Caco-2–specific rat LPH hypersensitivesite (rLHS) was identified in the region of the TATA box(rLHS1, Fig. 1B). At −78 bp, a hypersensitive site waspresent (rLHS2) that was more prominent in Caco-2 thanHep-G2 cells. In the region −164 to −138 bp, and in theregion between −226 to −200 bp, several rat LPH foot-prints (rLFP) and hypersensitive sites were identified(Fig. 1C), all of which were present in both Caco-2 andHep-G2 cells. Similarly, additional footprints were iden-tified in more distal regions (Fig. 1, D–F and data notshown).

To identify transcription factors that might bind tocandidate sequences displaying strong footprints, a com-puter analysis of the 2,038-bp LPH flanking sequencewas performed using the MatInspector program, whichallowed us to compare sites from the Transfac database(34). Because such analysis identifies numerous putativesites that bind transcription factors not known to be pres-ent in the intestine, additional analysis was focused ontranscription factors confirmed to be specifically ex-pressed by either hepatic or intestinal cells, or both.Table 1 lists these sites and candidate transcription fac-tors.

The region just 5� to the TATA box contained previ-ously identified sites present in human and pig LPH. Inaddition to the CE-LPHl (Cdx-2) site (16), a GATA sitewas identified in the human LPH proximal promoter (19)and an HNFl site was identified in the pig LPH promoter(21). GATA and HNFl sites in the proximal promoterwere also confirmed in the highly homologous rat LPHpromoter. These sites have been investigated in detailand published in studies from our laboratory (35). Ad-ditional investigation was focused on a possible repres-sor region, in which there was a potential site for theKruppel-like family of repressors, of which one member,formerly GKLF, now designated KLF4, is important inthe intestine. We also focused on potential binding sitesin the more distal flanking region for C/EBP andHNF3�, both of which are expressed in the endodermand regulate the expression of hepatocyte genes.

Deletion Analysis

By deletion analysis, we identified regions in the 5�-flanking sequence of the rat LPH gene that might play arole in the transcriptional control of cell-specific geneexpression and correlated such regions with potentialbinding sites identified by footprint analysis. We thentransiently transfected deletion constructs of the 5�-flanking sequence fused to hGH into LPH-expressingCaco-2 and Hep-G2 cells and, in some experiments, intoHeLa cells.

In these experiments, r37GH, which contained onlythe TATA box and sequence through the transcriptionalinitiation site, displayed low basal activity in bothCaco-2 and Hep-G2 cells (Fig. 2). When 74 bp of the ratLPH 5�-flanking sequence were included, transcriptionalactivity increased in both cell types with little differencebetween Caco-2 and Hep-G2 expression. In contrast,with 210 bp of the 5�-flanking sequence, transcriptionalactivity was reduced in both cell types as compared withr74GH. However, in Hep-G2 cells, r210GH was reducedto the level of basal transcription (r37GH), whereas inCaco-2 cells, r210GH remained approximately sixfoldgreater than basal transcription. There was little changein expression in either cell type as the length of thepromoter was increased from −210 to 1000 bp. Increas-

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ing the length of the promoter from 1000 to −2,038 re-vealed stepwise increases in both Caco-2 and Hep-G2expression. Constructs from −825 to −2,038 bp revealedan 8- to 16-fold greater expression in Caco-2 than Hep-G2 cells. The r2038GH construct, identical with the onedemonstrating enterocyte-specific expression in ourtransgenic studies (15), was the most active in Caco-2cells. When 4,000 bp of 5�-flanking sequence was used,expression in Caco-2 cells was reduced dramatically, butremained more than 50-fold greater than in HepG2 cells,whereas in the Hep-G2 cells, expression was nearly ex-tinguished and was below the level obtained with r37GH.These data suggest the presence of noncell line-specificenhancement caused by sequence between −74 and −37bp, cell-specific silencing attributed to sequence between−210 and −74 bp, and additional regulatory regions fur-ther 5� that eventually result in a highly cell line-specific(i.e., Caco-2 cell) response.

To analyze further the silencer region described inFigure 2, additional constructs were studied in Caco-2,

Hep-G2, and HeLa cells (Fig. 3). A binding site homolo-gous to that of Drosophila Kruppel was identified atrLHS2 (−78 bp, Table 1) in the silencer region of the ratLPH gene (−210 to −74 bp). Since Kruppel and Kruppel-like factors have been shown to repress gene transcrip-tion (36–38), additional transient transfection assayswere performed with a −95 hGH construct that encom-passed the Kruppel core binding site and with a constructthat contained a mutation in this site (r95mutGH) (Fig.3A). These experiments had two important results. First,the addition of the potential Kruppel-like repressor sitedid not reduce expression in Caco-2 or Hep-G2 cellsrelative to the −74 bp construct. Furthermore, inac-tivation of this site did not change transcriptional activityfrom that seen in the r95 wild type construct, suggest-ing that the putative Kruppel binding site plays no role inthe regulation of LPH expression. Although expressionwas less in the nonendodermal-derived HeLa cells, thelevel was the same as that of the −74 construct. Thus, therewas no indication of cell-specific repression at this site.

FIG. 1. DNase I footprint analysis of the rat LPH 5�-flanking region. (A) Map of footprinted regions. Probes are indicated by hatchedboxes, and the end that was labeled is indicated by an asterisk (*). Relevant restriction sites used in the preparation of probes areindicated. (B–F) Autoradiographs of the footprint gels. G, G-ladder; −, no protein; BSA, bovine serum albumin. Rat LPH hypersensitivesites (rLHS) and footprints (rLFP) are indicated on the right and the position of specific bases is indicated on the left. (B) Footprint assayof the proximal promoter region. (C–F) Footprint assays of sequence further 5�.

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Because the Kruppel-containing region demonstratedno detectable repression, the −210 to −95 bp region wasmapped further (Fig. 3B). Expression from r108GH andr133GH in Caco-2 and Hep-G2 cells was only slightlydifferent from levels displayed by r94GH and r74GH. Incontrast, r181GH showed markedly reduced expressionin both cell types, indicating possible silencing. Activityfrom r210GH was reduced further, but to a greater extentin Hep-G2 cells. In HeLa cells, transcriptional activityshowed a stepwise decrease as the length of 5�-flankingsequence was extended from −95 bp to −108 bp to −133bp. The activity of r133GH was near baseline, as was thatof r181 GH and r210GH. These data suggest a differen-tial silencer effect beginning in the −108 to −95 region inHeLa cells, but between −181 to −133 in Caco-2 andHep-G2 cells. Thus, it is possible that cell-specific tran-scription factors are responsible for silencing in thesedifferent cell types. The deletion analysis suggests thatthe region between −108 and −210 includes elementsconveying cell-specific regulation, as the expression inHeLa cells drops to baseline levels, while both Caco-2and Hep-G2 maintain expression. This region encom-passes two hypersensitive sites and two footprints (Table1 ). Sequence comparison indicated that one of these ispossibly a forkhead family binding site. Spodsberg et al.(21) identified binding of the forkhead-related proteinsFREAC-2 and FREAC-3 to a pig LPH region between

FIG. 2. Deletional analysis of the rat LPH 5�-flanking region inintestinal and nonintestinal cell lines. Deletions were made in ratLPH 5�-flanking sequence. The flanking regions fused to hGH(indicated on the left) were transiently transfected into Caco-2,Hep-G2, or HeLa cells, and hGH secreted into the media wasmeasured as described in Materials and Methods. All bars rep-resent at least three independent assays as described. All assayswere controlled for transfection efficiency and expressed relativeto the expression of pXGH5. The data are means (± SEM) foreach group.

TABLE 1. Correlation of DNase I footprint data with known transcription factor binding sites

Site* Location†Cell

specificity‡Transfac

match Comments

rLHS1 −35 C TBP TATA-binding protein site, critical for initiation of gene transcription(48,49).

rLHS2 −78 C>H Kruppel family Binding site for Drosophila Kruppel, role as repressor in segmentation(37) large family of related zinc-finger containing, repressor proteins

−82 HNF-1 Hepatocyte nuclear factor-1 site, role in expression of several intestinalgenes (50)

−90 GATA Transcription factors expressed in gut (51)rLFP1 −146 to −138 C,H No endoderm or

intestine-specificfactors

rLHS3 −149 C>H HNF3�/FREAC FOX family; several members demonstrated to be critical regulators ofendoderm differentiation (52)

C/EBP Family demonstrated to be critical regulators of hepatocyte andadipocyte genes (40)

rLHS4 −157 C,H no intestinal factorsrLFP3 −226 to −218 C,H HFH; GKLF; XFD2 Possible binding site FOX familyrLFP4 −471 to −466 C,H HNF3 FOX family; several members demonstrated to be critical regulators of

endoderm differentiationrLFP7 −570 to −553 C,H C/EBP CCAAT/enhancer binding protein site, role in cellular differentiation,

critical regulators of hepatocyte and adipocyte genes (40)rLFP11 −1346 to −1325 C,H HNF3 FOX family; several members demonstrated to be critical regulators of

endoderm differentiationrLFP12 −1926 to −1900 C,H HNF3, C/EBP FOX family; several members demonstrated to be critical regulators of

endoderm differentiation; CCAAT/enhancer binding protein site, rolein cellular differentiation, critical regulators of hepatocyte andadipocyte genes (40)

*r, rat;†Location is expressed relative to the transcriptional initiation site (+1).‡C, Caco-2.LHS, LPH hypersensitive sites; LFP, LPH footprint; H, Hep-G2.

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−227 and −299, which gave similar transcriptional re-pression in deletion analysis. Thus, although the pro-tein(s) that bind in this region of rat LPH are unidenti-fied, one of the forkhead family (now called FOX [fork-head box] (39)), which binds to elements similar to thosefor HNF3, may bind here. Additional studies to elucidatethese possibilities are under way in our laboratory.

Co-transfection Analysis

Comparison of results from deletion analysis andanalysis of footprints and hypersensitive sites identifiedthree possible binding sites for HNF3 (LFP4, LFP11, andLFP12) and one for C/EBP (LFP7) in regions that re-

duced reporter expression when deleted (Table 1).Analysis of LFP12 indicated possible binding of HNF3and C/EBP factors. Both LFP11 and LFP12 sequenceswere identical with the corresponding regions in the 5�-flanking region of mouse LPH (Montgomery et al.,manuscript in preparation), consistent with the hypoth-esis that they represent regulatory elements. Co-transfection experiments were performed to evaluate therole of C/EBP and HNF3 in regulation of rat LPH. Com-parison of HNF3� and � indicated that HNF3� gave agreater enhancement than did HNF3� (Fig. 4A). WhenHNF3� was co-transfected with the series of deletionconstructs, expression decreased with the deletion of thesegment between −2,038 and −1,520 (encompassingLFP12) and to a lesser degree between −1,820 and−1,000 (LFP11) and −1,000 to −825. Deletion from −825to −210 (LFP4) produced minimal change in expression.Finally, there was little difference in expression betweenthe −108 and −210 constructs, suggesting that HNF3� isnot the transcription factor binding in this region (Fig.4B). Removal of the sequence between −108 and −37dropped the enhancement of expression to baseline (Fig.4B). The three isoforms of C/EBP were tested, but onlyco-transfection of C/EBP� gave an enhancement of ex-pression with the −2,038 LPH construct in Caco-2 cells(data not shown). Computer analysis gave the strongfootprint at LFP7 the best math as a possible binding sitefor C/EBP. Therefore, the possible HNF3 sites LFP4,LFP11, and LFP12 and possible C/EBP site LFP7 werefurther investigated by EMSA.

EMSA Analysis

EMSA analysis focused on the four sites that seemedfrom prior analysis to have the greatest likelihood ofbinding C/EBP or HNF3. The putative C/EBP site atLFP7 bound a protein from Caco-2 cells as well as liverand HeLa cells. However, the complex could not be su-pershifted with antibodies against C/EBP isoforms, andthe protein was not heat stable, as is C/EBP, indicatingthat it is not C/EBP (40) (data not shown). BecauseC/EBP co-transfection enhances LPH reporter expres-sion, it likely acts through another as-yet-unidentifiedbinding site(s). EMSA of the putative HNF3� sites re-vealed that these sites specifically bound Caco-2 cellproteins (Fig. 5A, C). The identity of the proteins wasconfirmed by addition of antibodies against HNF3� andHNF3�. Both antibodies produced supershifts (Fig. 5B,C), indicating that both isoforms of the transcription fac-tor bound the site in vitro. This finding is consistent withreports that HNF3� and HNF3� are expressed in theintestine (41), as they are in Caco-2 cells (42). Since theco-transfection experiments showed that HNF� gavestronger enhancement of LPH expression, competitionfor binding may provide an additional regulatory mecha-nism determining the level of LPH expression in vivo.Thus, the co-transfection and EMSA data identify

FIG. 3. Localization of the cell-specific negative activity in the ratLPH 5�-flanking sequence. (A) Promoter-reporter constructs con-taining an additional deletion (r95GH) and a mutation(r95mutGH) introduced into the Kruppel (TAGATAACaacGT) re-gion. (B) Further deletional analysis of the −210 to −95 bp region.All constructs were transiently transfected into Caco-2, Hep-G2,and HeLa cells, as described in Materials and Methods. All as-says were controlled for transfection efficiency and expressedrelative to the expression of pXGH5. The data are means (±SEM)for each group. Data from Caco-2 cells in B were n = 2.

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HNF3� and HNF3ß as transcription factors binding tothe more distal 5� flanking region to enhance LPHexpression.

DISCUSSION

In this study we analyzed the cell-specific regulationof LPH expression using the first 2,038 bp of the rat LPH5�-flanking region that we (15) and others (17) havefound to contain the elements necessary for regulation ofin vivo expression. As shown in Figure 2, a 4-kb con-struct had an even higher degree of cell specificity, sug-gesting that other cell-specific elements may be presentin the additional 2 kb of the 5�-flanking region. Theirpotential function in vivo is not yet clear. A recent reporthas confirmed that transgenic mice with 2 kb of rat LPH5�-flanking region fused to a luciferase reporter dis-played cell-specific expression, and both regional anddevelopmental expression of the reporter that paralleledthat of the endogenous LPH gene, further indicating thatthe key regulatory elements are within the first 2 kb.These investigators found that an 800-bp construct gavelow levels of expression in multiple organs, suggestingthat there are critical distal elements between −800 bpand −2,038 bp necessary for accurate developmental and

tissue-specific expression (17), findings consistent withthose of the current study.

Key elements mediating endoderm-specific expres-sion are contained within the first 100 bases of the ratLPH 5�-flanking region, encompassing binding sites forCdx-2, GATA, and HNF1. Krasinski et al. (35) havepresented a detailed analysis of the interaction of thesefactors. The first 110 bp of the LPH 5�-flanking sequenceare highly conserved and contain transcription factor-binding sites that are virtually identical among the mam-mals in which this region has been analyzed. The func-tional significance of the HNF1 site for expression of pigLPH, and of the GATA site for human and rat LPH havebeen demonstrated using Caco-2 cells (19,21), but noprior data describe expression of LPH reporter constructsin nonintestinal, endodermally derived cells. The currentstudies using Hep-G2 and HeLa cells demonstrate LPHsilencing (Fig. 2). The presence of a repressor element inthe proximal promoter of rat LPH was also suggested bythe studies of Fang et al. (43).

Although we found no evidence for binding of aKruppel-like repressor at the indicated site, it remainspossible that a repressor binds in the region distal to theGATA site. Our findings suggest that cell-specific si-lencing occurs in the rat gene in a region between −210

FIG. 4. (A) Comparison of effects of HNF3� and HNF3ß on LPH expression. Co-transfection of short, intermediate, and long promoter-reporter constructs previously described with expression vectors for HNF3� and HNF�, as well as control vector (PRC-CMV) into Caco-2cells. (B) Effects of HNF3ß co-transfection on promoter-reporter constructs. Co-transfection of previously described series of promoter-reporter constructs from −2,038 to −37 bp with expression vector for HNF�, as well as control vector (PRC-CMV) into Caco-2 cells.

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FIG. 5. (Figure legend on facing page)

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and −95 bp. Sequences further 5� increase the cell-specific response by increasing expression in Caco-2cells and decreasing expression in Hep-G2 cells. The−210 to −95 region displays two footprints common toCaco-2 and Hep-G2 cells, and a common and a Caco-2cell-specific hypersensitive site. For both sucrase-isomaltase and IFABP, two carefully studied intestinalgenes, repression appears to be an important mechanismof intestine-specific gene expression (44). Although fur-ther characterization will be necessary to completely de-fine the factors involved in these mechanisms of regula-tion, it appears that gene repression is important in thecell-specific control of LPH gene expression.

Our data indicate that there are additional positiveregulators in the more distal region of the LPH promoter,between −2,038 and −1,000. The LPH 5�-flanking regioncontains several possible C/EBP and HNF3 binding sites,identified by sequence analysis of regions protected byprotein binding in our footprint analysis. Both C/EBPand HNF3 have been demonstrated to enhance the ex-pression of pig LPH constructs (21). Our co-transfectionexperiments indicate that C/EBPa enhanced LPH expres-sion, but the most likely binding site was ruled out. Fur-ther analysis will be required to determine where C/EBPdoes bind. In our experiments, binding of HNF3 to LFP4,LFP11, and LFP12 was demonstrated by EMSA. How-ever, LFP4 is located in a region that appears to be littleaffected in co-transfection assays. In contrast, LFP11 islocated between −1,000 and −1,580, in which a moderateeffect of HNF3 is observed, whereas the region between−1,580 and −2,038 encompassing LFP12 displays astronger effect of HNF3. The high degree of sequenceconservation of these regions in mouse and rat is con-sistent with a functional role.

Comparisons between the LPH 5�-flanking regions ofrodents and humans or humans and pigs has identifiedseveral putative transcription factor-binding sites, butlittle sequence similarity beyond the proximal promoter(21). Thus, the emerging pattern suggests a highly con-served proximal LPH promoter, whose function is modu-lated by more diverse distal elements in different mam-mals.

LPH levels in the enterocyte are known to be regulatedby corticoid and thyroid hormones (45). Thus, it is of

interest that no response element for either hormone hasbeen identified in the rat LPH 5�-flanking region, indi-cating that the gene is not directly regulated by thesehormones. Similarly, none has been reported in the hu-man LPH gene (46). Although unidentified response el-ements may be present more distally, the observed hor-monal effects may be mediated via intermediary factorsor by mesenchymal cells, as suggested by the experi-ments Kedinger et al. (47).

To date, the only intestine-specific transcription factoridentified as a regulator of LPH is Cdx-2. HNF1, GATA,HNF3, and C/EBP are all found in hepatocytes as well asother tissues. If Cdx-2 is critical to enterocyte-specificexpression, it must act in concert with other factors be-cause the proximal promoter in which its binding site liesdoes not convey cell-specific expression in our experi-ments. There may be other as-yet-unidentified Cdx-2sites, as indicated in pig LPH (21). Another possiblepromoter mechanism is differential expression of tran-scription factors and their isoforms either during devel-opment or along the proximal/distal or crypt/villus axes.The three isoforms of HNF3 are expressed at somewhatdifferent times in development, and their tissue expres-sion in the intestinal tract differs (41). Our co-transfection data suggest that HNF3� is more effectivethan HNF3� in enhancing LPH in vitro, whereas of thethree C/EBP isoforms, only C/EBP� appears to enhanceLPH expression. These data are consistent with our find-ing that C/EBP� protein was the only isoform detectablein fetal enterocytes (22). Similar results for HNF1� andHNF1�, as well as C/EBP, in pig LPH were presented bySpodsberg et al. (21). It is unknown whether changes inisoform expression also occur in vivo. It is likely that thelevel of LPH transcription is a result of the combinedeffects of transcription factors expressed in cell, region,and development-specific patterns.

These data suggest that repression is an important partof the intestine-specific regulation of rat lactase-phlorizin hydrolase, in combination with positive regu-latory elements in the highly conserved proximal pro-moter. HNF3� enhances LPH expression, and threeHNF3� sites in the LPH 5�-flanking region have beenidentified. Two of these are in regions whose deletionreduces the response to co-transfection, suggesting that

<FIG. 5. (A) EMSA of putative HNF3 sites LFP4 and LFP11. As described in Materials and Methods, synthetic oligomers encompassingeach of the two putative HNF3 sites were labeled and run alone (Lanes 1 and 7), or incubated with Caco-2 proteins (Lanes 2–6, 8–12).Proteins were incubated with no competitor DNA (Lanes 2 and 8), with increasing amounts of unlabeled specific competitor DNA beforeaddition of labeled probe (Lanes 3–5 and 9–11), and with unlabeled nonspecific competitor (Lanes 6 and 12). (B) Supershift EMSA ofputative HNF3 sites LFP4 and LFP11. As described in Materials and Methods, synthetic oligomers encompassing the two putative HNF3sites were labeled and incubated with Caco-2 proteins (Lanes 2 and 7). Proteins were incubated with unlabeled specific competitor DNAbefore addition of labeled probe (Lanes 3 and 8). Antibodies against HNF3� (Lanes 4 and 9) and HNF3ß (Lanes 5 and 10) were addedto the incubation before gel analysis. Finally, an antibody to the unrelated transcription factor HNF6 was added to an identical protein/DNAcomplex (Lanes 6 and 12). Sc, specific complex; ss, supershifted complex. (C) EMSA of putative HNF3 site LFP12. As described inMaterials and Methods, a synthetic oligomer encompassing the putative HNF3 and C/EBP sites was labeled and incubated with Caco-2proteins (Lanes 2–10). Proteins were incubated without competitor (Lane 2), with increasing amounts of unlabeled specific competitorDNA before addition of labeled probe (Lanes 3–5), and with an unlabeled nonspecific competitor (Lane 6). Antibodies (5 µL) againstHNF3� (Lane 7) and HNF3� (Lane 8) and C/EBP� (Lane 9) were added to the incubation before gel analysis. As an additional control,an equal volume (5 µL) of nonimmune serum was added to the protein/DNA complex (Lane 10).

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they are functionally important. Although binding sitesremain to be identified, C/EBP� also enhances LPH ex-pression.

As noted, these studies were performed in vitro usingthe Caco-2 and Hep-G2 cell lines as model s stems. Suchdata usually, but not always, reflect the function of smallintestinal and liver cells. Confirmation ultimately willrequire in vivo models. Additional experiments, includ-ing site-directed mutagenesis, will be required to com-pletely delineate which specific sites and factors are bothnecessary and sufficient to determine intestinal LPH ex-pression.

Acknowledgments: The authors thank Drs. A. Leiter, D.Jefferson, and A. Kane, Directors of the Molecular BiologyCore, Cell Culture Core, and Mocrobiology Core (respec-tively), of the Center for Gastroenterology Research on Ab-sorptive and Secretory Processes (GRASP), P30DK34928,Boston, Massachusetts, U.S.A., for their support and advice.The authors also thank Rebecca M. June and Ketu Mishra fortheir work on the project, as well as Linda Rogers, Moira Brett,Elisa Moy, Willem Louis, and Leah Moyer for their help withcell culture. The authors thank Dr. Steven McKnight, Univer-sity of Texas Southwestern Medical Center, for the generousgift of C/EBP expression vectors, and Dr. Robert Costa, Uni-versity of Illinois Medical College, Chicago, Illinois, U.S.A.,for the generous gift of HNF3 expression vectors and antibod-ies against HNF3� and �.

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